Cardiac Overexpression of a Gq Inhibitor Blocks Induction of Extracellular Signal–Regulated Kinase and c-Jun NH2-Terminal Kinase Activity in In Vivo Pressure Overload
Background—Understanding the cellular signals that initiate cardiac hypertrophy is of critical importance in identifying the pathways that mediate heart failure. The family of mitogen-activated protein kinases (MAPKs), including the extracellular signal–regulated kinases (ERKs), c-Jun NH2-terminal kinase (JNK), and p38 MAPKs, may play specific roles in myocardial growth and function.
Methods and Results—To determine the mechanism of activation of MAPK pathways during the development of cardiac hypertrophy, we evaluated the induction of MAPK activity after aortic constriction in wild-type and in 2 types of cardiac gene-targeted mice: one overexpressing a carboxyl-terminal peptide of Gαq that inhibits Gq-mediated signaling (TG GqI mouse) and another overexpressing a carboxyl-terminal peptide of β-adrenergic receptor kinase-1 that inhibits Gβγ signaling (TG βARKct mouse). Wild-type mice with pressure overload showed an acute induction of JNK, followed by the induction of p38/p38β at 3 days and ERK at 7 days. Both JNK and p38 activity remained elevated at 7 days after banding. In TG GqI mice, hypertrophy was significantly attenuated, and induction of ERK and JNK activity was abolished, whereas the induction of p38 and p38β was robust, but delayed. By contrast, all 3 MAPK pathways were activated by aortic constriction in the TG βARKct hearts, suggesting a role for Gαq, but not Gβγ.
Conclusions—Taken together, these data show that the induction of ERK and JNK activity in in vivo pressure-overload hypertrophy is mediated through the stimulation of Gq-coupled receptors and that non–Gq-mediated pathways are recruited to activate p38 and p38β.
Although cardiac hypertrophy is thought to be an adaptive response of the heart to a variety of stimuli, it is associated with an increased mortality and increased incidence of heart failure in epidemiological studies.1 2 Understanding the cellular signals that initiate the hypertrophic response is of critical importance in identifying pathways that mediate this maladaptive deterioration. In vitro studies have suggested a pivotal role of Gq-coupled receptor signaling in promoting cardiomyocyte hypertrophy.3 4 Furthermore, cardiac overexpression of Gq-coupled receptors or Gαq itself in transgenic mice leads to myocardial hypertrophy,5 6 apoptosis,6 7 and heart failure.6 7
G protein–coupled receptors (GPCRs) are able to activate mitogen-activated protein kinases (MAPKs) and, under certain conditions, will lead to a mitogenic response.8 9 The MAPK superfamily includes 3 major pathways: the extracellular signal–regulated kinase (ERK)1/2 pathway and 2 stress-activated protein kinase pathways, c-Jun NH2-terminal kinase (JNK) and p38 MAPK.10 Activation of MAPK pathways by growth factors, cytokines, and cell stress selectively mediates a variety of cellular responses ranging from cell growth and differentiation to apoptosis. Studies by Xia et al11 in rat PC-12 pheochromocytoma cells have demonstrated that the dynamic balance between growth factor–activated ERK and stress-activated JNK-p38 pathways may determine whether a cell survives or undergoes apoptosis.
In cell culture studies, it has recently been reported that although signaling through different G proteins (Gq, Gi, and Gs) can selectively activate MAPKs and promote cell differentiation, stimulation of only Gq-coupled receptors can equally activate all 3 major MAPK pathways.12 Interestingly, in cultured rat neonatal cardiomyocytes, 2 isoforms of p38 kinase, p38 and p38β, appear to have distinct functions13 : the p38β isoform promotes a hypertrophic phenotype, and the p38 isoform tends to promote an apoptotic phenotype.13 Furthermore, the induction of ERK activity in cultured cardiac fibroblasts was found to be mediated by Gβγ signals, whereas in cultured cardiac myocytes, it was Gq-mediated.14
The important role of GPCRs and MAPK signaling in the development of cardiac hypertrophy has recently been shown by the generation of transgenic mice overexpressing Gαq6 15 and the angiotensin type 1 receptor6 15 and by the adenovirus-mediated transfer of a dominant inhibitory mutant of an upstream activator of JNK.16 Furthermore, overexpression of a constitutively active mutant of the transforming growth factor-β–activated kinase, a member of the MAPK kinase kinase family, leads to cardiac hypertrophy and dysfunction in transgenic mice.17 However, whether Gq-coupled receptor stimulation is required for the induction of MAPK pathways in in vivo hypertrophy remains unclear. In this regard, we have recently demonstrated that inhibition of Gq-coupled receptor signaling in transgenic mice significantly reduces the hypertrophic response to in vivo pressure overload.18 Inhibition of Gq signaling was achieved through overexpression of a carboxyl-terminal peptide of Gαq that inhibits the heterotrimeric Gq interaction with agonist-occupied receptors. Although that study showed a critical role for Gq signals in mediating the hypertrophic phenotype, it did not identify the downstream cellular pathways involved.
To determine whether the mechanism for the induction of MAPK in cardiac hypertrophy is dependent on Gq- and/or Gβγ-mediated pathways, we evaluated ERK1/2, p38, p38β, and JNK activity during the development of in vivo pressure overload in wild-type mice, Gq inhibitor transgenic mice (TG GqI), and transgenic mice overexpressing a peptide inhibitor of Gβγ signaling (TG βARKct, where βARK indicates β-adrenergic receptor kinase).
Adult wild-type and transgenic mice, inbred on a C57/B6 background, were studied at 2 to 3 months of age. The transgenic mice used in the present study were (1) mice with cardiac-specific overexpression of a carboxyl-terminal peptide of Gαq that inhibits Gq-mediated signaling (TG GqI mice)18 and (2) transgenic mice with cardiac-targeted overexpression of a peptide inhibitor of Gβγ-mediated signaling (TG βARKct mice).19 The βARKct peptide is composed of the last 194 amino acid residues of βARK1 and contains the domain responsible for Gβγ binding, a process required for βARK1 activation.19 The cardiac phenotypes of the TG GqI and TG βARKct mice were previously described for 2 independent lines,18 19 which have remained consistent over numerous generations. The animals in the present study were handled according to approved protocols and animal welfare regulations by the Institutional Review Board at Duke University Medical Center.
Transthoracic 2D guided M-mode echocardiography was performed in anesthetized mice (2.5% Avertin, 14 μL/g IP) before and 7 days after the induction of pressure-overload hypertrophy, with use of an HDI 5000 echocardiograph (Advanced Technology Laboratories) as previously described.20 Parameters measured are shown in the Table⇓.
In Vivo Pressure-Overload Hypertrophy
Mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (2.5 mg/kg), and transverse aortic constriction (TAC) was performed as previously described.21 Sham-operated mice underwent the same operation except for aortic constriction.
At 7 hours, 3 days, or 7 days after surgery, sham-operated and banded mice from either the wild-type, TG GqI, or TG βARKct groups were anesthetized and intubated, and after bilateral vagotomy the trans-stenotic gradient was assessed by recording the simultaneous measurement of right and left carotid artery pressures. Hearts were then excised, and chambers were dissected free, weighed, and then frozen in liquid N2 within 25 seconds from harvesting. Despite identical surgical techniques, a broad range in the ratio of left ventricular (LV) weight to body weight (LVW/BW) is found after TAC, which varies directly with the level of systolic pressure gradient.18 Therefore, to avoid experimental bias, hearts for the MAPK assay from all groups were chosen from animals with a trans-stenotic pressure gradient between 45 and 100 mm Hg, thereby eliminating the high and low extremes.
Immunodetection of myocardial levels of MAPKs was performed on cytosolic extracts from LVs after immunoprecipitation using polyclonal antibodies to total ERK2-p42/ERK1-p44, p38, p38β, and JNK1-p46/JNK3 (Santa Cruz Biotechnology). The kinases were detected with secondary antibodies conjugated with horseradish peroxidase (ECL, Amersham Pharmacia Biotech).
MAPK assay was performed as previously described.18 Briefly, 2 mg of clarified LV extract in 2 mL of RIPA (150 mmol/L NaCl, 50 mmol/L Tris-Cl [pH 8.0], 5 mmol/L EDTA [pH 8.0], 1% v/v Nonidet P-40, 0.5% w/v deoxycholate, 10 mmol/L NaF, 10 mmol/L sodium pyrophosphate, 100 mmol/L phenylmethylsulfonyl fluoride, 2 μg/mL aprotinin, and 2 μg/mL leopeptin) was immunoprecipitated at 4°C for 2 hours with the use of antibodies to ERK2-p42/ERK1-p44, p38, p38β, and JNK1-p46/JNK3 (Santa Cruz Biotechnology) and protein A–agarose or protein G–agarose (Boehringer-Mannheim). The immunoprecipitates were pelleted and washed twice with 1 mL of RIPA and twice with 1 mL of kinase assay buffer. Samples were then resuspended in 40 μL of kinase buffer with 20 μmol/L ATP, [γ-32P]ATP (20 μCi/mL), and myelin basic protein (0.25 mg/mL) or glutathione S-transferase (GST)-c-Jun (10 μg) and incubated at 30°C for 20 minutes.
Reactions were terminated by adding 40 μL of 2× Laemmli loading buffer, and 30 μL of each reaction was electrophoresed through a 15% polyacrylamide/Tris-glycine gel. Phosphorylated myelin basic protein and GST-c-Jun on dried gels were quantified with a PhosphorImager (Molecular Dynamics).
Data are expressed as mean±SEM. One-way ANOVA was used to evaluate the echocardiographic measurements, heart weight, and kinase activity data before and after aortic constriction and among wild-type, TG GqI, and TG βARKct mice. Post hoc testing was performed with a Scheffé test. For all analyses, a value of P<0.05 was considered significant.
Physiological Response to In Vivo Pressure Overload in Wild-Type and TG GqI Mice
To evaluate the hypertrophic response after TAC, we measured LVW/BW in wild-type and TG GqI mice at 7 hours, 3 days, and 7 days after surgical pressure overload (Figure 1⇓ and Table⇑). Three days after TAC, wild-type mice developed a small but nonsignificant 16% increase in LVW/BW compared with sham-operated mice. Seven days after TAC, wild-type mice developed significant LV hypertrophy with a 54% increase LVW/BW compared with sham-operated mice. No hypertrophy was detected at 7 hours after TAC.
In contrast, 3 days after TAC, TG GqI mice developed a minimal 5% increase in LVW/BW compared with that in sham-operated mice (Figure 1⇑, P=NS). Similar to our previous result,18 the banded TG GqI mice developed a significantly blunted increase in LVW/BW compared with that in wild-type mice (22% versus 54%, respectively, P<0.001; Table⇑ and Figure 1⇑) despite a similar trans-stenotic pressure gradient 7 days after TAC (74.4±3.6 mm Hg; Table⇑ and Figure 1⇑). No hypertrophy was detected at 7 hours after TAC. Echocardiography in TG GqI mice showed no change in LV size or percent fractional shortening after TAC, suggesting the preservation of cardiac function despite the blunted hypertrophic response (Table⇑). Postsurgical mortality in banded TG GqI mice was not different from that in wild-type mice (19% and 20%, respectively).
Induction of MAPK Activity With In Vivo Pressure Overload in Wild-Type and TG GqI Mice
We evaluated JNK, ERK, p38, and p38β activity 7 hours, 3 days, and 7 days after aortic constriction in wild-type and TG GqI mice. Data are represented as fold induction in TAC hearts compared with sham-operated hearts for each of the groups.
A significant induction of JNK activity, compared with that after sham surgery, was observed during the initial phase of pressure overload, as early as 7 hours after TAC, showing the sensitive nature of JNK activation to acute stress (Figure 2a⇓ and 2b⇓). Interestingly, compared with JNK activity after the sham operation, JNK activity remained elevated after 3 days and 7 days of pressure overload during the period of early cardiac hypertrophy to established cardiac hypertrophy (Figure 2a⇓ and 2b⇓). To determine whether Gq-mediated pathways are involved in the induction of MAPK activity, we measured the time course of JNK activity in TG GqI mice after TAC. As shown in Figure 2c⇓ and 2d⇓, the induction of JNK activity was completely abolished after acute pressure overload (7 hours) and remained abolished up to 7 days after pressure overload in the hearts of TG GqI mice.
The pattern of ERK activity after pressure overload in wild-type mice differed from the pattern of JNK activity, as shown in Figure 3a⇓ and 3b⇓. Compared with the corresponding sham-operated hearts, hearts from wild-type mice showed a small nonsignificant (1.3-fold) increase in ERK activity at 3 days of TAC that became markedly increased after 7 days of pressure overload. Similar to the pattern observed for JNK, the banded TG GqI mice showed that the induction of ERK activity was completely blocked early (3 days) and later (7 days) after TAC (Figure 3c⇓ and 3d⇓).
p38 and p38β Activity
In wild-type mice, a different pattern of activation for p38 and p38β by pressure overload was observed compared with that seen for JNK and ERK. In this case, strong induction of p38 and p38β activity was seen as early as 3 days, which remained elevated at 7 days. In contrast, p38 and p38β were significantly activated in wild-type hearts compared with sham-operated TG GqI hearts, but only after 7 days of in vivo pressure overload (Figure 4b⇓ and 4d⇓).
In all extracts examined, no significant differences were found in total MAPK protein levels between sham operation and TAC for both wild-type and TG GqI mice, as assessed by Western blot, suggesting only a modulation of kinase activities after TAC (data not shown).
MAPK Activation in TG βARKct Mice
To determine whether a mechanism for the induction of MAPK activity in cardiac hypertrophy involves Gβγ subunits released from either Gq-coupled receptors or from other GPCRs, experiments were performed in TG βARKct mice. The βγ subunits of G proteins (Gβγ) have been shown to activate signaling pathways in a variety of cells,22 23 including phosphoinositide 3-kinase (PI3K) in in vivo pressure-overload hypertrophy.24 Thus, we tested whether activation of MAPK in hypertrophied hearts involves Gβγ. As shown in the Table⇑, banded TG βARKct mice develop cardiac hypertrophy in response to pressure overload to the same level as found in wild-type mice.
Heart extracts from sham-operated and 7-day banded TG βARKct mice were used to measure MAPK activity. As shown in Figure 5⇓, a statistically significant increase in activity for all the MAPKs tested was observed in the banded TG βARKct hearts at 7 days after TAC compared with sham-operated TG βARKct hearts. Importantly, the level of induction of all 3 MAPK pathways in the TG βARKct TAC hearts was similar to that seen in the wild-type TAC hearts. Taken together, these data suggest a role for Gαq, but not Gβγ, in the activation of MAPK pathways with pressure-overload cardiac hypertrophy.
Basal MAPK activity was evaluated in heart extracts from sham-operated wild-type, TG GqI, and TG βARKct mice (n=5 for each group). No significant difference was found in the basal ERK, JNK, or p38 (α or β) activity among the 3 groups.
In the present study, we report the time course of induction of all 3 major MAPK pathways (ERK, JNK, and p38) with the development of in vivo cardiac hypertrophy and establish the essential role for Gq signaling in this process. We show that there are 3 phases to the induction of MAPK activity during in vivo hypertrophy. In the first phase (acute pressure overload, 7 hours), only JNK activity is increased. The second phase (early cardiac hypertrophy, 3 days) is associated with robust induction of p38, p38β, and JNK activity but with a minimal increase in ERK activity. The final phase (established hypertrophy, 7 days) is associated with strong and sustained induction of all the MAPK pathways. This pattern of MAPK activation is markedly altered in the pressure-overloaded TG GqI mice. In this case, JNK and ERK activity are completely abolished, whereas p38 and p38β activation occurs but is delayed in appearance, suggesting recruitment of other non–Gq-mediated pathways for p38 activation.
In cultured cells, activation of the MAPKs is thought to be mediated by both α and βγ subunits of the heterotrimeric G protein after GPCR stimulation.25 26 27 28 However, among α subunits, only Gαq stimulation has been shown to activate all MAPK pathways.12 In the rat, Yano et al29 have recently shown that angiotensin II–induced cardiac hypertrophy is associated only with the early and transient activation of JNK. Although the study by Yano et al suggests that stimulation of the JNK pathway can induce a hypertrophic response, it did not assess the mechanism for activation in response to in vivo pressure overload. In the present study, we used the physiological stimulus of pressure overload and show a specific pattern of MAPK activation with early and persistent JNK activation, followed by both p38 and p38β and then ERK. Furthermore, we show that all MAPKs are activated by signals originating from Gq-coupled receptors and that later recruitment of non–Gq-coupled receptor pathways can eventually lead to p38 activation once hypertrophy is established. Finally, it is possible that the strong induction in p38 MAPK activity in the TG GqI mice, which we show is non–G-protein (Gαq or Gβγ)–mediated, is responsible for the mild hypertrophy that develops in these animals with pressure overload.
A recent study has reported that inhibition of JNK in the heart by expression of a dominant inhibitory activator mutant can inhibit the development of cardiac hypertrophy after banding in the rat.16 The present study adds to those findings by showing that the development of cardiac hypertrophy is associated with the induction of all 3 MAPK pathways and that the increase in activity is mediated through Gq-coupled receptors. Furthermore, the late induction of p38 and p38β MAPK activity in banded TG GqI mice suggests that non–Gq-mediated pathways or signaling cascades can be recruited to activate p38 in the pressure-overloaded heart.30 The non–Gq-mediated induction of p38 is consistent with a recent study by Zhang et al17 showing that overexpression of a constitutively active mutant of transforming growth factor-β–activated kinase, a mediator of transforming growth factor-β signaling, results in the activation of p38 but not ERK or JNK MAPK. Also consistent with our findings is the in vitro study by Sabri et al31 showing that in mouse cardiomyocytes, p38 MAPK activation was coupled to β-adrenergic but not α1-adrenergic receptor stimulation.
We have previously shown that in TG βARKct mice, Gβγ dimers released from stimulation of Gq-coupled receptors can activate PI3K24 and that PI3K can activate MAPK.32 Therefore, we tested whether released Gβγ subunits from either Gq receptors or other GPCRs play a role in the increase in MAPK activity with hypertrophy. We studied MAPK activity 7 days after TAC in mice overexpressing the βARKct, and we have determined that JNK activity and ERK activity are significantly increased in hearts from TG βARKct mice. Although we show that Gq-coupled receptor stimulation is required for the induction of JNK and ERK activity, by performing TAC in the TG βARKct mice, we also demonstrate that Gβγ subunits arising from Gq heterotrimers (or for that matter from any other GPCR) do not significantly contribute to the induction of the 3 MAPK pathways. Nonetheless, because the βARKct transgene is driven by the myosin heavy chain promoter, we cannot exclude a role for cardiac fibroblasts in the induction of MAPK activity in cardiac hypertrophy.14
This work was supported in part by National Institutes of Health grants HL-56687 (Dr Rockman) and HL-61690 (Dr Koch). We gratefully acknowledge Debbie Colpitts for her expert secretarial assistance.
- Received July 21, 2000.
- Revision received September 14, 2000.
- Accepted September 19, 2000.
- Copyright © 2001 by American Heart Association
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